Research Paper Volume 14, Issue 15 pp 6202—6226

Figure 5. SIAH1 is an E3 ligase to induce RPS3 ubiquitination. (A–F) HEK293T cells were transfected plasmids as indicated for 36 h. Cell lysates were subjected to denatured immunoprecipitation and Western blotting. Cells were treated with MG132 (10 μM) for 6 h before cell lysis. (A) SIAH1 overexpression induced ubiquitination and degradation of endogenous RPS3. (B) SIAH1 overexpression induced ubiquitination and degradation of exogenous RPS3. (C) When SIAH1 was knocked down, the ubiquitination level of RPS3 was significantly reduced. (D) Upon SIAH1 expression, FLAG-RPS3 (K214R) had a significantly lower ubiquitination than the wild type (FLAG-RPS3). (E) SIAH1 KD, induced no significant decrease in the ubiquitination level of FLAG-RPS3 (K214R). (F) In SIAH1-inhibited A2780 cells, mutation of the RING finger domain of SIAH1 significantly reduced the ubiquitination level of RPS3.